This invention relates to the detection of the presence and concentration of unbound species, e.g., hormones, in a liquid sample such as serum. More particularly, it relates to the detection of free species of the type which are capable of reversibly binding with protein in samples containing the free species, the binding protein, and a concentration of species-protein complex.
The "competitive assay" technique for measuring the concentration of various biologically active substances for diagnostic purposes is now well established. The technique involves a reaction system wherein antibody specific to the species to be determined is incubated together with the test sample and an aliquot of a distinguishable analogue of the sample. The analogue and natural species compete for sites of attachment to the antibody, and after separation of the antibody from the remainder of the reaction system, the antibody or supernatant may be assayed for analogue. The amount of analogue associated with the antibody is an inverse function of the concentration of the test species in the sample.
Hormones such as those excreted from the thyroid, tests, ovaries, adrenal cortex, and other mammalian glands are often transported through the circulatory system in association with hormone binding proteins or globulins. Often, it is of diagnostic significance to be able to determine the presence and concentration of free hormone, as opposed to protein-bound hormone or total hormone. For example, thyroxine (T.sub.4) in the bloodstream exists in a dynamic equilibrium as a species bound to a transport protein, (thyroxine-binding globulin, albumin, or pre-albumin) and, in a small fraction (potentially 0.1% or less), as an unbound substance ("free" or unbound T.sub.4). Free T.sub.4 is thought to be the active species of the thyroid hormone system. Unfortunately, free-T.sub.4 assay methodology has been tedious, complicated, expensive, and error-prone because of the difficulty in standardization of materials and in measuring minute quantities of unbound hormone in the presence of overwhelming amounts of hormone relatively loosely bound to protein. The competitive assay procedure noted above is not capable of determining free species concentrations.
Of the methods for measuring free T.sub.4 and other hormones which exist in vivo as protein complexes, the dialysis method affords a high degree of accuracy. A dialysis bag containing known quantities of serum to be tested and labled hormone is suspended in buffer. The labled hormone distributes itself between the free and bound states in the same proportions as the serum hormone. The mass of labled hormone added must be extremely small so as to avoid seriously perturbing the system, yet the count rate must be high so that detection of small amounts is feasible. In the case of radioactive lables, this requires that labled hormone of very high specific activity be used. After a suitable period (about 24 hours), the tagged and natural hormone not bound to protein diffuse through the semipermeable dialysis bag while hormone bound to protein (molecular weight greater than 20,000) remains within the dialysis bag. To determine the quantity of free hormone present in the serum, one assays an aliquot of the buffer for labled hormone. As a result of the assay, the fraction of labled hormone that crossed into the buffer may be calculated, and the fraction of total hormone that exists in the free state inferred. To convert this fraction to mass units (e.g., ng/dl) of free hormone, a total hormone assay must also be run on the sample.
While this system can give meaningful results in free T.sub.4 and other free hormone assays, it is poorly suited for routine use. Two tests must be run, and the accuracy of the final result can be compromised by either procedure. Rather large amounts of radioactivity must be employed per test. Interfering impurities in the labeled hormone can cause special problems, and long incubation times are required. Only serum can be used, and it must be extremely fresh. Furthermore, the collection and standardization of all the materials necessary for the assay is not a routine matter. Thus, the application of the procedure has been limited due to the high cost and labor intensity of the method and the skilled personnel required.
Another method of free hormone assay involves reaction kinetics and requires two separate tests. Each test measures a kinetic curve related to how fast an antibody captures hormone, e.g.., T.sub.4, away from the opposing pull of the primary binder such as thyroxine binding globulin.
In such an assay system, inaccuracies result from several pathologic conditions. If more protein binding sites are present than usual, the effective attraction of the globulin for the hormone will be greater and the rate of binding to antibody will decrease. In the case of thyroxine analysis, this would give the appearance that the sample had little T.sub.4 when in fact there might be a high level of T.sub.4, but all bound.